Piezo1-mitochondrial calcium uniporter coupling-dependent mechanotransduction basis for acupuncture to drive analgesia

Abstract Background The mechanosensitive ion channel Piezo1 is a critical sensor of mechanical forces in diverse biological contexts, albeit its role in translating the acupuncture-induced mechanical stimulation into analgesic signaling remains largely unexplored. Acupuncture is a widely used therapy for pain management, with needle manipulation-generated mechanical forces considered central to its therapeutic efficacy. However, the molecular mechanisms underlying this mechanotransduction process remain unknown. Here, we identified myocyte Piezo1 at the ST36 acupoint as a key initiator converting local needling force into peripherally derived analgesic signaling under complete Freund’s adjuvant (CFA)-induced inflammatory pain conditions. Methods In mice with CFA-induced inflammatory pain, we assessed the analgesic efficacy of deep needling at ST36 using paw withdrawal latency tests. The underlying molecular mechanisms were examined through immunohistochemistry, immunofluorescence, western blotting, transmission electron microscopy, enzyme-linked immunosorbent assay, electromyography, and in vivo two-photon calcium (Ca) imaging. To evaluate the Piezo1-Ca 2 ⁺-calmodulin (CaM)-Ca 2 ⁺/CaM-dependent protein kinase II (CaMKII)-signaling axis and its downstream effects on mitochondrial function and purinergic signaling, we employed muscle-specific adeno-associated virus-mediated knockdown of Piezo1 and CaM together with a panel of pharmacological modulators (GsMTx4, Yoda1, TFP, 2-APB, RU360, DPCPX). Results We first confirmed Piezo1 expression in myocytes within the ST36 acupoint, and muscle-specific Piezo1 knockdown abrogated the analgesic effects of deep needling at ST36. Mechanical stimulation by needling activated Piezo1, thereby triggering Ca 2 ⁺ influx, increasing CaM expression and CaMKII phosphorylation, and enhancing sarcoplasmic reticulum-mitochondrial coupling via the inositol trisphosphate receptor-1 (IP3R1)-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel-1 (VDAC1) complex and mitochondrial Ca uniporter (MCU) pathway. These events promoted mitochondrial Ca 2 ⁺ uptake and increased adenosine triphosphate (ATP)/adenosine production. Both genetic and pharmacological inhibition of Piezo1, CaM, or downstream signaling molecules blocked the analgesic effects of acupuncture, whereas direct Piezo1 activation with Yoda1 mimicked these effects. In vivo two-photon Ca imaging confirmed that electroacupuncture-evoked myocyte Ca 2 ⁺ transients are Piezo1-dependent, and the resulting adenosine may mediate remote analgesia through the activation of the adenosine A1 receptor. Conclusion Piezo1 acts as a core peripheral mechanotransducer that converts local mechanical stimuli generated by acupuncture into downstream analgesic signaling in CFA-induced inflammatory pain. This study delineates a specific myocyte-originated mechanotransduction axis underlying acupuncture analgesia under inflammatory conditions, thereby providing mechanistic insights for understanding acupoint-specific therapeutic effects.